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Use of cyclophilin as antioxidant and prevention of cyclosporin a-induced toxicity in cell transplantation by overexpression of cyclophilin

Use of cyclophilin as antioxidant and prevention of cyclosporin a-induced toxicity in cell transplantation by overexpression of cyclophilin description/claims

This application is a divisional of U.S. patent application Ser. No. 10/635,264 filed Aug. 6, 2003, which in turn claims the benefit of priority from Korean Patent Application No. 10-2002-0069822 filed Nov. 11, 2002, the contents of each of which are incorporated herein by reference.

1. Field of the Invention

The present invention relates to the use of cyclophilin with peptidyl-propyl-cis-trans isomerase (PPIase) activity as an antioxidant and a method of preventing immunosuppressant cyclosporin A (CsA)-induced toxicity in cell transplantation by overexpressing cyclophilin.

2. Brief Description of the Prior Art

CsA (Cyclosporin A) is a potent immunosuppressant that is widely used in organ transplantation and autoimmune diseases (Alejandro, D. S., et al., J. Am. Soc. Nephrol. 5, 153-160, 1994). CsA is a cyclic undecapeptide that bind to cyclophilin A (CypA) with a high affinity. CypA is a cytosolic protein with PPIase (peptidyl-propyl-cis-trans isomerase) activity that is potently inhibited by CsA binding. PPIase enzymes function as molecular chaperones to faciliate protein folding, intracellular trafficking and maintenance multi-protein complex stability (Andreeva, L., et al., Int. J. Exp. Pathol. 80, 305-315, 1999; Hamilton, G. S., et al., J. Med. Chem. 41, 5119-5143, 1998), although it is believed that inhibition of PPIase activity is not required for its immunosuppressive action (Bierer, B. E., et al., Science 250, 556-559, 1990). CsA-CypA complex, but not CypA alone, binds and inhibits the activity of calcineurin, which is a calcium/calmodulin-dependent protein phosphates. (Friedman, J., et al., Cell 66, 799-806, 1991; Liu, J., et al., Biochemistry 31, 3896-3901, 1992). The inhibitor of calcineurin activity blocks the translocation of NFATs (nuclear factors of activated T-cells), which in turn prevents T-helper cells from expressing several lymphokines that mediate the activation of immune reaction (Matsuda, S., et al., Immunopharmacology 47, 119-125, 2000).

Although CsA made an exceptional turning point in transplantation and autoimmune disease treatment, its toxicity in various tissues including skeletal muscle caused the patients serious troubles (Rush, D. N., Clin. Biochem. 24, 101-105, 1990; Arellano, F., et al., Lancet 337, 915, 1991; Biring, M. S., et al., J. Appl. Physiol. 84, 1967-1975, 1998). CsA-induced myopathies clinically cause myalgia, cramps, muscle weakness and elevation of plasma creatine kinase. (Goy, J. J., et al., Lancet 1, 1446-1447, 1989; Fernadex-Sola, J., et al., Lancet 335, 362-363, 1990). There have been many attempts to reveal the mechanism of CsA-induced myopathy. Several groups have proposed that CsA hinders muscle regeneration from satellite myoblasts since CsA was shown to induce muscle deficiency in regenerated muscle fibers, and to inhibit myogenic differentiation in cultured myoblasts (Hardiman, O., et al., Neurology 43, 1432-1434, 1993; Abbott, K. I., et al., Mol. Biol. Cell 9, 2905-2916, 1998; Friday, B. B., et al., J. Cell Biol. 149, 657-666, 2000). Other studies revealed the activities of CsA to decreases mitochondrial respiration (Hokanson, J. F., et al., Am. J. Respir. Crit. Care. Med. 151, 1848-1851, 1995) and to inhibit creatine uptake as a result of creatine transporter reduction (Tran, T. T., et al., J. Biol. Chem. 275, 35708-35714, 2000), suggesting that CsA-induced myopathies might be related to mitochondrial injury or altered energy states.

Myoblasts or satellite cells represent a population of myogenic stem cells that can proliferate and fuse to or replace damaged muscle myofibers. Using dy/dy and mdx mouse models of genetic myopathies, it was shown in the late 1980's that normal genes could be introduced into dystrophic muscles of genetic myopatheis (Law, P. K. et al., Muscle Nerve 11, 525-533, 1998; Partridge, T. A. et al., Nature 337, 176-179, 1989). On the basis of this finding, allogenic myoblast transplantation (AMT) was tried in patients suffering from Duchenne dystrophy, typical of severe hereditary muscle disorders. Unfortunately, the initial clinical trials were unsuccessful (Skuk, D. et al., Microsc. Res. Tech. 48, 213-222, 2000). Since then, many research groups took up the study of improving the survival rate of transplanted myoblasts using animal models. One of the results obtained is that transplanted myoblasts are primarily rejected by immune response (Gill, R. G. et al., Cell Transplant 4, 361-370, 1995). Thus, it was found that the success of AMT needs a suitable immunosuppressant to be administered prior to transplanting myoblasts.

Known as immunosuppressants useful for the transplantation of myoblasts are CsA, tacrolimus (FK506) and cyclophosphamide. FK506 showed a success rate of 95% or higher both in clinical trials and animal model studies (Kinoshita, I. et al., Muscle Nerve 17, 1407-1415, 1994; Kinoshita, I. et al., Muscle Nerve 18, 1217-1218, 1995), while cyclophosphamide did not increase the survival of the fused muscle fibers after AMT (Karpati, G. et al., Ann. Neurol. 34, 8-17, 1993; Vilquin, J. T. et al., Neuromuscul. Disord. 5, 511-517, 1995), and CsA ensuresd only a moderate success rate for the transplantation (Labrecque, C. et al., Transplant. Proc. 24, 2889-92, 1992).

Although CsA is a potent immunosuppressant marking a turning point in transplantation, it has been used with great reluctance due to its low success rate associated with the following problems. CsA causes cytotoxicity in various types of cells through reactive oxygen species (ROS) (Wang, C. et al., Transplantation 58, 940-946, 1994; Perez de Lema, G. et al., Life Sci. 62, 1745-1753, 1998; Wolf, A. et al., J. Pharmacol. Exp. Ther. 280, 1328-1334, 1997). Further, CsA is found to give rise to muscle depletion in regenerated muscle fibers and inhibit muscle differentiation in myoblast cultures, thereby blocking muscle regeneration from satellite myoblasts (Hardiman, O. et al., Neurology 43, 1432-1434, 1993; Abbott, K. L. et al., Mol. Biol. Cell, 9, 2905-2916, 1998; Friday, B. B. et al., J. Cell Biol., 149, 657-666, 2000).

With the aim of raising transplantation success rates, attempts have been made to remove CsA-induced toxicity. For instance, antioxidants were used together with CsA (Wang, C. et al., Transplantation 58, 940-946, 1994; Perez de Lema, G. et al., Life Sci. 62, 1745-1753, 1998; Kumar, K. V. et al., Transplantation 67, 1065-1068, 1999; Naidu, M. U. et al., Nephron. 81, 60-66, 1999). Antioxidants could partially protect cells from the apoptosis attributed to intracellular ROS increase, but could not prevent the blockage of cell differentiation.

Accordingly, there remains a need for better alleviating the cellular toxicity induced by CsA so as to improve the success rate of cell transplantation.

The thorough and intensive research into the alleviation of CsA-induced toxicity, conducted by the present inventors, resulted in the finding that the CsA-induced toxic effect on transplanted cells, that is, the apoptosis and cell differentiation blockage of transplanted cells, is caused by oxidative stress, at least partly via inhibition of the PPIase activity of CypA. Indicating that the PPIase activity of CypA is directly involved in cell differentiation, this finding is in contrast to the conventional assertion that the CsA-induced cell differentiation blockage results from the inhibition of calcineurin activity. Also, the present inventors found that the cells that have survived after pre-exposure to CsA could not only reversibly proliferate and differentiate, but also are resistant to subsequent CsA exposure. On the basis of these findings, the present inventors established that the CsA-induced cytotoxicity effect on transplanted cells can be remarkably reduced by the overexpression of CypA in the cells to be transplanted.

In accordance with an aspect of the present invention, there is provided an antioxidant, comprising a cyclophilin protein with PPIase activity.

In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for preventing cyclosporin A-induced cytotoxicity by the overexpression of cylcophilin with PPIase activity in the transplanted cells, comprising a recombinant expression vector which can express the cyclophilin protein in such a sufficient amount as to reduce the toxicity induced by cyclosporin A or its analogues.

In accordance with a further aspect of the present invention, there is provided a cell for use in the transplantation which is resistant to cyclosporin A or its analogues, wherein a cyclophilin protein with PPIase activity is over-expressed.

In accordance with still another aspect of the present invention, there is provided a method of preparing the cells for use in the transplantation which is resistant to cyclosporin A or its analogues, comprising the steps of introducing a gene encoding a cyclophilin protein with PPIase activity into a vector to construct a recombinant expression vector, transfecting the recombinant expression vector into the cells for transplantation, culturing the transfected cells, and selecting cells in which the cyclophilin with PPIase activity is over-expressed.

In accordance with still a further aspect of the present invention, there is provided a method of preparing the cells for use in the transplantation which are resistant to cyclosporin A or its analogues, comprising the steps of culturing the cells for transplantation in the presence of cyclosporin A or its analogues and recovering viable cells from the cultures.

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